E.coli Endonuclease VIII functions as both an N-glycosylase (by excising oxidative base lesions) and an AP lyase (by subsequently cleaving the phosphodiester backbone), leaving terminal phosphates at the 5′ and 3′ ends. (1) Damaged bases removed by Endonuclease VIII include: urea, 5, 6- dihydroxythymine, thymine glycol, 5-hydroxy-5- methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea (2,3).
Source of Protein
An E. coli strain which carries the cloned Endonuclease VIII gene.
10 mM Tris-HCl
250 mM NaCl
0.1 mM EDTA
pH 8.0 @ 25°C
B9080 10X Endonulease Vlll Buffer
10X Endonuclease Vlll Buffer (B9080)
100 mM Tris-HCl
750 mM NaCl
10 mM EDTA
pH 8.0 @ 25°C
One unit is defined as the amount of enzyme required to cleave 1 pmol of an oligonucleotide duplex containing a single AP site in 1 hour at 37°C.
|Storage Temperature||-25 to -15°C|
|Specific Activity||770,000 U/mg|
|SS Exonuclease||10 U <1.0% released|
|DS Exonuclease||100 U <1.0% released|
|E.coli DNA Contamination||100 U <10 copies|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
QUALITY CONTROL ANALYSIS
Unit Characterization Assay
Specific activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were prepared in Endo VIII glycerol storage solution and added to 10 µL reactions containing 2.0 µM of an FAM-labeled, 34-base, duplex oligonucleotide, containing a single Uracil. [Note: substrate pre-treated for 2 minutes with 1 unit of UDG to create an abasic site] Reactions were incubated 60 minutes at 37°C, plunged on ice, denatured with N-N-dimethylformamide and analyzed by running and exposing to short-wave UV a 15% TBE-Urea acrylamide gel.
SDS-Page (Physical Purity Assessment) and Specifications
2.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.
Single-Stranded Exonuclease Activity
A 50 µL reaction containing 15,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µL reaction containing 15,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.
E.coli 16S rDNA Contamination Test
Replicate 5 µL samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.